Synthetic biological system construction and green intelligent biological manufacturing

The production of chemicals in microbial cell factories is one of the effective ways to solve energy and environmental problems. Many chemical products have been produced by synthetic biology, but production ability and robustness of the strains still needs to been improved. Development of intelligent cell factory and realizing intelligent biological manufacturing is an important approach to improve the production and robustness. This review introduces the research status of intelligent biological manufacturing from five following aspects: protein design, biosensor, metabolism regulation, strain evolution and fermentation process. The development of biological “intelligence” will make an important contribution to improving the production level of industrial biological processes and process energy saving and emission reduction.     

Flow-injection amperometric glucose biosensors based on graphene/Nafion hybrid electrodes

In this research, we demonstrated the fabrication of flow-injection amperometric glucose biosensors based on RGO/Nafion hybrids. The nanohybridization of the reduced graphene oxide (RGO) by Nafion provided the fast electron transfer (ET) for the sensitive amperometric biosensor platforms. The ET rate (k_s) and the charge transfer resistance (R_CT) of GOx-RGO/Nafion hybrids were evaluated to verify the accelerated ET. Moreover, hybrid biosensors revealed a quasi-reversible and surface controlled process, as confirmed by the low peak-to-peak (ΔE_p) and linear relations between I_p and scan rate (ν). Hybrid biosensors showed the fast response time of ∼3 s, the sensitivity of 3.8 μA mM⁻¹ cm⁻², the limit of detection of 170 μM, and the linear detection range of 2–20 mM for the flow-injection amperometric detection of glucose. Furthermore, interference effect of oxidizable species such as ascorbic acid (AA) and uric acid (UA) on the performance of hybrid biosensors was prevented at the operating potential of −0.20 V even under the flow injection mode. Therefore, the fast, sensitive, and stable amperometric responses of hybrid biosensors in the flow injection system make it highly suitable for automatically monitoring glucose.     

Direct electron transfer from glucose oxidase immobilized on a nano-porous glassy carbon electrode

A pair of well-defined and reversible redox peaks was observed for the direct electron transfer (DET) reaction of an immobilized glucose oxidase (GOx) on the surface of a nano-porous glassy carbon electrode at the formal potential (E°′) of −0.439 V versus Ag/AgCl/saturated KCl. The electron transfer rate constant (k_s) was calculated to be 5.27 s⁻¹. The dependence of E°′ on pH indicated that the direct electron transfer of the GOx was a two-electron transfer process, coupled with two-proton transfer. The results clearly demonstrate that the nano-porous glassy carbon electrode is a cost-effective and ready-to-use scaffold for the fabrication of a glucose biosensor.     

Poly(glycidyl methacrylate‐co‐3‐thienyl‐methylmethacrylate) based working electrodes for hydrogen peroxide biosensing

BACKGROUND: Hydrogen peroxide biosensors based on Poly(glycidyl methacrylate‐co‐3‐thienylmethylmethacrylate)/ Polypyrrole [Poly(GMA‐co‐MTM)/PPy] composite film were reported. Poly(GMA‐co‐MTM) including various amounts of GMA and MTM monomers was synthesized via the radical polymerization. Enzyme horseradish peroxidase (HRP) was trapped in Poly(GMA‐co‐MTM)/PPy composites during the electropolymerization reaction between pyrrole and thiophene groups of MTM monomer, and chemically bonded via the epoxy groups of GMA. Analytical parameters of the fabricated electrodes were calculated and are discussed in terms of film electroactivity and mass transfer conditions of the composite films. RESULTS: The amount of electroactive HRP was found to be 1.25, 0.34 and 0.213 µg for the working electrodes of Poly(GMA_30%‐ co‐MTM_70%)/PPy/HRP, Poly(GMA_85%‐co‐MTM_15%)/PPy/HRP and Poly(GMA_90%‐co‐MTM_10%)/PPy/HRP, respectively. Optimal response of the fabricated electrodes was obtained at pH 7 and an operational potential of ? 0.35 V. It was observed that effective enzyme immobilization and electroactivity of the composite films could be changed by changing the ratios of GMA and MTM fractions of Poly(GMA‐co‐MTM) based working electrodes. CONCLUSION: The amount of electroactive enzyme increases with increasing MTM content of the final copolymer. High operational stabilities of the biosensors can be attributed to the strong covalent enzyme linkage via the epoxy groups of GMA due to preventing enzyme deterioration and loss. A more convenient microenvironment for mass transfer was provided for the electrodes by higher GMA ratios. It is observed that mass transfer is dominated by the mechanism of electron transfer to obtain effective sensitivity values. This work contributes to discussions clarifying the problems regarding the design parameters of biosensors. Copyright © 2011 Society of Chemical Industry     

Synthesis and Characterization of Sulfonated Polyanilines and Application in Construction of a Diazinon Biosensor

A series of sulfonated polyaniline/derivatized polyaniline nanocomposites was chemically synthesized in the presence of anthracene and naphthalene sulfonic acids. UV-vis and FTIR results indicated the emergence of new bands at 420 and 700 nm and 1100 cm^?1 in their spectra, respectively, meaning the dopant/polymer intercalations involved electronic interactions between dopant/polymer sub-lattices. The electroactive composites displayed moderately fast electrode kinetics. A composite-based biosensor for hydrogen peroxide reached a steady state current of 9.2 and 5.3 µA, and when fine-tuned for diazinon detection gave % inhibitions to be 41 and 81% for the polyaniline and poly-o-methoxyaniline biosensors, respectively.     

Amperometric Cholesterol Biosensor Using Layer-by-Layer Adsorption Technique on Polyaniline-coated Polyester Films

An amperometric cholesterol biosensor was fabricated using polyaniline-coated polyester films. Polyaniline was dissolved in chloroform with camphorsulfonic acid, and polystyrene was added to this solution. Using this mixed solution, the coating was placed onto polyester films. Cholesterol oxidase was immobilized onto these films using an electrostatic layer-by-layer adsorption technique. Poly(diallyldimethylammonium chloride) was used as the counter ion source. The level of adsorption was examined and evidence of layer-by-layer adsorption was investigated using a quartz crystal microbalance (QCM). A cholesterol biosensor was fabricated from these films as a working electrode, and it was used to measure the cholesterol concentration.     

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.     

Hf-ZnO酪氨酸酶生物传感器检测邻苯二酚

以四氯化铪(HfCl4)和硝酸锌为原料,通过水热法制得Hf掺杂氧化锌纳米材料(Hf-ZnO),并将Hf-ZnO、酪氨酸酶(Tyr)和壳聚糖(CS)修饰在玻碳电极(GCE)上,制得Tyr/Hf-ZnO/CS/GCE生物电极。利用FESEM、DLS、XRD和XPS对Hf-ZnO的结构和性能进行表征。采用循环伏安伏安法(CV)和计时电流法(IT)对Tyr/Hf-ZnO/CS/GCE电极进行电化学测试。结果表明,Tyr/Hf-ZnO/CS/GCE电极在pH 5和-50 mV的低电势下对邻苯二酚有最佳检测能力,对邻苯二酚检测的线性范围是0.5~47 μmol/L,灵敏度为195 μA/(mmol/L),检测限是0.1215 μmol/L(S/N=3)。此外,该生物电极的稳定性和重复性好,可有效避免尿素、多巴胺、抗坏血酸等与邻苯二酚电活性相近物质的干扰。     

静电纺丝法制备ZnO构建酪氨酸酶生物传感器检测邻苯二酚

电化学生物传感器在环境监测、生物与食品分析等领域应用广泛。本文采用静电纺丝法制备了氧化锌微纳米纤维材料,并负载酪氨酸酶（Tyr）构建了检测邻苯二酚的生物传感电极。利用X射线粉末衍射（XRD）、扫描电子显微镜（SEM）表征了氧化锌微纳米纤维的形貌结构,采用循环伏安法（CV）和计时电流法（IT）优化了检测邻苯二酚的工作条件。结果表明,制备的Tyr/ZnO/CS/GCE生物传感电极具有良好的电化学性能,在反应过程中为扩散控制,对邻苯二酚的检测在5～50μmol/L的浓度范围内具有较好的线性关系,最低检测限为1.9041μmol/L,灵敏度为376.31μA/(mmol·L·cm²),氧化锌的加入可增强酪氨酸酶的稳定性,在尿素、多巴胺和葡萄糖3种电化学活性相近物质存在的情况下仍对邻苯二酚的检测有较好的选择性,且具有良好的循环稳定性。